基于甘草次酸的脂质纳米粒可高效递送mRNA  

Glycyrrhetinic acid⁃based lipid nanoparticles efficiently deliver mRNA

作　　者：路海涛 麦洁锐 曹艺明 桑野 苗明三[1] 杨静[2] LU Haitao;MAI Jierui;CAO Yiming;SANG Ye;MIAO Mingsan;YANG Jing(Henan University of Chinese Medicine,Zhengzhou 450046,China;Institute of Microbiology Epidemiology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)

机构地区：[1]河南中医药大学,郑州450046 [2]军事科学院军事医学研究院微生物流行病研究所,北京100850

出　　处：《军事医学》2023年第11期835-843,共9页Military Medical Sciences

基　　金：国家重点研发计划(2021YFC2302405)。

摘　　要：目的为避免主流脂质纳米粒(LNP)递送载体中胆固醇带来的潜在危害,采用甘草次酸完全替代胆固醇的方法,设计并制备了基于甘草次酸的脂质纳米粒(GA⁃LNP),并综合评价了该脂质纳米粒作为mRNA递送载体的潜力。方法制备基于甘草次酸的有机相溶液,通过微流控纳米药物合成仪包封mRNA形成GA⁃LNP/mRNA复合物,测定复合物的粒径、Zeta电位、多分散系数(PDI)及稳定性;通过透射电子显微镜(TEM)拍摄其形态;进行细胞转染及小鼠活体成像验证GA⁃LNP的体内外递送效果;通过递送G⁃CSF mRNA进一步验证了递送载体的性质、体内衰减时间及安全性,对递送载体进行综合评价。结果GA⁃LNP/eGFP mRNA复合物、GA⁃LNP/G⁃CSF mRNA复合物平均粒径分别为(96±2.1)nm、(85.2±2.1)nm,平均电位分别为(11.6±1.6)mV、(6.4±2.9)mV,形态呈均匀球形。GA⁃LNP具备在细胞及动物体内高效递送报告基因mRNA和G⁃CSF mRNA的效果,体内表达的G⁃CSF蛋白浓度可高于正常值4 d以上,未出现小鼠生化指标及病理切片显著性变化,且致炎性弱于LNP复合物。结论制备的基于甘草次酸的新型脂质纳米粒,具有高效递送mRNA的能力,复合物稳定性、安全性较好,可为制备无胆固醇参与的mRNA递送载体提供新思路。Objective To prepare glycyrrhetinic acid⁃lipid nanoparticles(GA⁃LNPs)and evaluate their potential as mRNA delivery vectors.Methods Firstly,organic phase solution based on glycyrrhetinic acid was prepared beforethe reporter gene mRNA was encapsulated by the microfluidic nano drug synthesizer to form GA⁃LNP/mRNA complexes.The particle size,Zeta potential,polydispersity(PDI),and stability of the complexes were measured,whose morphology was photographed with the transmission electron microscope(TEM).The in vitro and in vivo delivery of GA⁃LNP was verified by cell transfection and in vivo imaging of mice.By delivering G⁃CSF mRNA,the properties,decay time,and safety of delivery vectors were explored so that these vectors were comprehensively evaluated.Results The average particle size of GA⁃LNPs/eGFP mRNA complexes and GA⁃LNPs/G⁃CSF mRNA complexes was(96±2.1)nm and(85.2±2.1)nm,respectively,and the average potential was(11.6±1.6)mV and(6.4±2.9)mV,respectively.Their shapes were uniform and spherical.The GA⁃LNPs were capable of efficiently delivering reporter gene mRNA and G⁃CSF mRNA in cells and animals.The concentration of G⁃CSF protein expressed in vivo remained higher than the normal value for over four days,and no significant changes in mouse biochemical indexes or pathological sections were observed.The inflammatory effect was weaker than that of LNP complexes.Conclusion Using glycyrrhetinic acid,a novel type of lipid nanoparticle has been prepared,which can efficiently deliver mRNA.GA⁃LNP/mRNA complexes are highly stable and safe,which can provide a new perspective on the preparation of cholesterol⁃free mRNA delivery vectors.

关 键 词：甘草次酸 胆固醇 脂质纳米粒 mRNA递送 G⁃CSF mRNA 

分 类 号：R284[医药卫生—中药学] Q522[医药卫生—中医学]